Nucleic acid encoding eosinophil eotaxin receptor

ABSTRACT

The eosinophil eotaxin receptor has been isolated, cloned and sequenced. This receptor is a human β-chemokine receptor and has been designated “CC CKR3”. The eosinophil eotaxin receptor may be used to screen and identify compounds that bind to the eosinophil eotaxin receptor. Such compounds would be useful in the treatment and prevention of atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) fromprovisional application Case Number 19634PV, filed Apr. 26, 1996 andfrom provisional application Case Number 19697PV, filed Apr. 26, 1996 asU.S. Ser. No. 60/016,158.

FIELD OF THE INVENTION

This invention relates to an eosinophil eotaxin receptor (“CC CKR3”), inparticular, the human eosinophil eotaxin receptor and nucleic acidsencoding this receptor. This invention further relates to assays whichmay be used to screen and identify compounds that bind to the eosinophileotaxin receptor. Such compounds would be useful in the treatment andprevention of atopic conditions including allergic rhinitis, dermatitis,conjunctivitis, and particularly bronchial asthma.

BACKGROUND OF THE INVENTION

Eosinophils play prominent roles in a variety of atopic conditionsincluding allergic rhinitis, dermatitis, conjunctivitis, andparticularly bronchial asthma (for a reviews see e.g. Gleich, G. J., etal., Eosinophils. J. I. Gallin, I. M. Goldstein, R. Snyderman, Eds.,Inflammation: Basic Principles and Clinical Correlates (Raven Press,Ltd., New York, 1992) and Seminario, M. C., et al. (1994) CurrentOpinion in Immunology 6, 860-864). A pivotal event in the process is theaccumulation of eosinophils at the involved sites. While a number of theclassical chemoattractants, including C5a, LTB4, and PAF, are known toattract eosinophils (Gleich, G. J., et al., Eosinophils. J. I. Gallin,et al. Eds., Inflammation: Basic Principles and Clinical Correlates(Raven Press, Ltd., New York, 1992)), these mediators are promiscuous,acting on a variety of leukocytes including neutrophils, and areunlikely to be responsible for the selective accumulation ofeosinophils. In contrast, the chemokines a family of 8-10 kDa proteinsare more restricted in the leukocyte subtypes they target and arepotential candidates for the recruitment of eosinophils in atopicdiseases and asthma (Baggiolini, M., Dewald, B. and Moser, B.(1994)Advances in Immunology 55, 97-179). Although there is a mountingbody of evidence that eosinophils are recruited to sites of allergicinflammation by a number of β-chemokines, particularly eotaxin andRANTES, the receptor which mediates these actions has not beenidentified.

The chemokines contain four conserved cysteines, and are divided intotwo sub-families based on the arrangement of the first cysteine pair(Baggiolini, M., Dewald, B. and Moser, B. (1994) Advances in Immunology55, 97-179). In the α-chemokine family, which includes IL-8, MGSA, NAP-2and IP-10, these two cysteines are separated by a single amino acid,while in the β-chemokine family, which includes RANTES (“regulated onactivation T expressed and secreted”), MCP-1 (“monocyte chemotacticprotein”), MCP-2, MCP-3, MIP-1α (“macrophage inflammatory protein”),MIP-1β and eotaxin, these two cysteines are adjacent. There is afunctional correlate to this structural division. The α-chemokines actprimarily on neutrophils, and the β-chemokines on monocytes,lymphocytes, basophils and eosinophils (Baggiolini, M., Dewald, B. andMoser, B. (1994) Advances in Immunology 55, 97-179). In particular,RANTES, MCP-2, MCP-3, and MIP-1α have been shown to activate eosinophilsin vitro (Dahinden, C. A., et al. (1994) Journal of ExperimentalMedicine 179, 751-756; Ebisawa, M., et al. (1994) Journal of Immunology153, 2153-2160; Weber, M., et al. (1995) Journal of Immunology 154,4166-4172), and RANTES to selectively attract eosinophils in vivo(Meurer, R., et al. (1993) Journal of Experimental Medicine 178,1913-1921; Beck, L., et al. (1995) FASEB Journal 9, A804). Similarly,eotaxin, a new member of the 13-chemokine family, first described inguinea pigs (Griffiths-Johnson, D. A., et al. (1993) Bichemical andBiophysical Research Communications 197, 1167-1172; Jose, P. J., et al.(1994) Journal of Experimental Medicine 179, 881-887) and mice(Rothenberg, et al. (1995) Proceedings of the National Academy ofSciences 92, 8960-8964) is also a potent attractant and activator ofeosinophils both in vitro and in vivo. Moreover, eotaxin is generatedduring antigenic challenge in the guinea pig model of allergic airwayinflammation (Jose, et al. (1994) J. Exp. Med., 179, 881-887;Rothenberg, et al. (1995) J. Exp. Med., 181, 1211-1216. The cloning ofguinea pig eotaxin has been disclosed (PCT Patent Publication No. WO95/07985; Mar. 23, 1995). The cloning of the human eosinophilchemoattractant eotaxin has recently been reported (Ponath, et al., J.Clin. Invest. (1996) 97(3) 604-612) and eotaxin has been suggested to bea very important agent in the mechanism of allergic inflammation(Baggiolini, et al., J. Clin. Invest. (1996) 97(3) 587).

Eosinophils are attracted by a number of β-chemokines, the most potentof which are eotaxin (Griffiths-Johnson, D. A., et al. (1993) Bichemicaland Biophysical Research Communications 197, 1167-1172; Jose, P. J., etal. (1994) Journal of Experimental Medicine 179, 881-887; Rothenberg, etal. (1995) Proceedings of the National Academy of Sciences 92,8960-8964) and RANTES (Dahinden, C. A., et al. (1994) Journal ofExperimental Medicine 179, 751-756; Ebisawa, M., et al. (1994) Journalof Immunology 153, 2153-2160; Weber, M., et al. (1995) Journal ofImmunology 154, 4166-4172; Meurer, R., et al. (1993) Journal ofExperimental Medicine 178, 1913-1921; Beck, L., et al. (1995) FASEBJournal 9, A804). Although several human β-chemokine receptors have beencharacterized in detail, none have the appropriate selectivity toaccount for the observed responses.

While elucidation of the actions of β-chemokines on eosinophils hascontributed greatly to the understanding of eosinophil biology,information regarding the cell surface receptors which mediate theseeffects remain sparse. Furthermore, there are no reports describingbinding studies of any of the β-chemokines to primary eosinophils. Theknown β-chemokine receptors are members of the G protein-coupledreceptor superfamily. Two of these receptors, CC CKR1 (12, 13) and CCCKR2 (MCP-1R) (Charo, I. F., et al. (1994) Proceecing of the NationalAcademy of Sciences 91, 2752-2756; Myers, S. J., et al. (1995) Journalof Biological Chemistry 270, 5786-5792; Franci, C., et al. (1995)Journal of Immunology 154, 6511-6517) found on monocytes, have beenextensively studied and their selectivity for the different chemokinesdefined. However, neither of these receptors has the necessary ligandselectivity or the appropriate expression patterns required to mediatethe effects of the β-chemokines on eosinophils. For example, CC CKR1binds RANTES with high affinity, but binds eotaxin poorly, and while theeffects of eotaxin on CC CKR2 have not been studied this receptor has noavidity for RANTES (Myers, S. J., et al. (1995) Journal of BiologicalChemistry 270, 5786-5792).

A review of the role of chemokines in allergic inflammation is providedby Kita, H., et al., J. Exp. Med. 183, 2421-2426 (June 1996). Inparticular, this review discusses the role which the receptor CKR-3plays in the process of allergic inflammation. The cloning, expressionand characterization of the human eosinophil eotaxin receptor has beenreported by Daugherty, B., J. Exp. Med. 183, 2349-2354 (May 1996). Thispublication discloses the cloning and functional expression of thechemokine receptor CC CKR3, as well as its characterization.

The cloning and expression of a human eosinophil receptor was allegedlyachieved by Combadiere, C., et al., J. Biological Chem. 270 (27),16491-16494 (Jul. 14, 1995). However, in a subsequent retraction (J.Biological Chem. 270, 30235 (1995)) they confirmed that the receptorwhich was actually cloned and expressed was not CC CKR3, but was anotherCC chemokine receptor CC CKR5. This receptor was subsequentlycharacterized by Kitaura, M., et al., J. Biological Chem. 271 (13),7725-7730 (Mar. 29, 1996).

A human eotaxin receptor has been reported by Ponath, P. D., et al. J.Exp. Med. 183, 2437-2448 (June 1996) and Gerard, C. J., et al., PCTPublication No. WO 96/22371 (Jul. 25, 1996). However, the sequencedisclosed in this publication possesses an error in the assignment ofthreonine rather than serine at position # 276 of the receptor. Inaddition, functionality of the receptor was not fully demonstrated.

A retrovirus designated human immunodeficiency virus (HIV-1) is theetiological agent of the complex disease that includes progressivedestruction of the immune system (acquired immune deficiency syndrome;AIDS) and degeneration of the central and peripheral nervous system.This virus was previously known as LAV, HTLV-III, or ARV. Entry of HIV-1into a target cell requires cell-surface CD4 and additional host cellcofactors. Fusin has been identified as a cofactor required forinfection with virus adapted for growth in transformed T-cells, however,fusin does not promote entry of macrophagetropic viruses which arebelieved to be the key pathogenic strains of HIV in vivo. It hasrecently been recognized that for efficient entry into target cells,human immunodeficiency viruses require the chemokine receptors CCR-5 andCXCR-4, as well as the primary receptor CD4 (Levy, N. Engl. J. Med.,335(20), 1528-1530 (Nov. 14 1996). The principal cofactor for entrymediated by the envelope glycoproteins of primary macrophage-trophicstrains of HIV-1 is CCR5, a receptor for the β-chemokines RANTES, MIP-1αand MIP-1β (Deng, et al., Nature, 381, 661-666 (1996)). HIV attaches tothe CD4 molecule on cells through a region of its envelope protein,gp120. It is believed that the CD-4 binding site on the gp120 of HIVinteracts with the CD4 molecule on the cell surface, and undergoesconformational changes which allow it to bind to another cell-surfacereceptor, such as CCR5 and/or CXCR-4. This brings the viral envelopecloser to the cell surface and allows interaction between gp41 on theviral envelope and a fusion domain on the cell surface, fusion with thecell membrane, and entry of the viral core into the cell. It has beenshown that β-chemokine ligands prevent HIV-1 from fusing with the cell(Dragic, et al., Nature, 381, 667-673 (1996)). It has further beendemonstrated that a complex of gp120 and soluble CD4 interactsspecifically with CCR-5 and inhibits the binding of the natural CCR-5ligands MIP-1α and MIP-1β (Wu, et al., Nature, 384, 179-183 (1996);Trkola, et al., Nature, 384, 184-187 (1996)).

Humans who are homozygous for mutant CCR-5 receptors which do not serveas co-receptors for HIV-1 in vitro apper to be unusually resistant toHIV-1 infection and are not immunocompromised by the presence of thisgenetic variant (Nature, 382, 722-725 (1996)). Absence of CCR-5 appearsto confer protection from HIV-1 infection (Nature, 382, 668-669 (1996)).Other chemokine receptors may be used by some strains of HIV-1 or may befavored by non-sexual routes of transmission. Although most HIV-1isolates studied to date utilize CCR-5 or fusin, some can use both aswell as the related CCR-2B and CCR-3 as co-receptors (Nature Medicine,2(11), 1240-1243 (1996)). The determination that chemokine receptors arecritical co-receptors for the entry of HIV into cells was pronounced a“1996 Breakthrough of the Year” by Science Magazine (Science, 274,1987-1991 (Dec. 20, 1996)).

The use of orally-active agents which modulate the action of theeosinophil eotaxin receptor would be a significant advance in thetreatment and prevention of atopic conditions including allergicrhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma.Further, agents which could block the eosinophil eotaxin receptor inhumans who possess normal chemokine receptors should prevent infectionin healthy individuals and slow or halt viral progression in infectedpatients.

It would also be desirable to know the molecular structure of theeosinophil eotaxin receptor in order to analyze this new receptor familyand understand its normal physiological role. This could lead to abetter understanding of the in vivo processes which occur uponligand-receptor binding. Further, it would be desirable to usecloned-eosinophil eotaxin receptor as essential components of an assaysystem which can identify new agents for the treatment and prevention ofatopic conditions.

SUMMARY OF THE INVENTION

The present invention relates to a novel receptor which is theeosinophil eotaxin receptor. This receptor is a human β-chemokinereceptor and has been designated “CC CKR3”. One aspect of the presentinvention is directed to the human eosinophil eotaxin receptor, freefrom receptor-associated proteins. A further aspect of this invention isthe human eosinophil eotaxin receptor which is isolated or purified.

Another aspect of this invention are eosinophil eotaxin receptors whichare encoded by substantially the same nucleic acid sequences, but whichhave undergone changes in splicing or other RNA processing-derivedmodifications or mutagenesis induced changes, so that the expressedprotein has a homologous, but different amino acid sequence from thenative forms. These variant forms may have different and/or additionalfunctions in human and animal physiology or in vitro in cell basedassays.

The present invention further provides the eosinophil eotaxin receptor,CC CKR3, which is a 13-chemokine receptor and which was cloned fromprimary eosinophils, and expressed in AML14.3D10 cells. This receptorbinds the potent eosinophil attractants, eotaxin, RANTES and MCP-3 withhigh affinity. In addition, eotaxin and RANTES, and to a lessor extentMCP-3, induce Ca²⁺-fluxes in cells expressing CC CKR3. Correlation withthe binding properties of primary eosinophils provide conclusiveevidence that CC CKR3 is the primary endogenous receptor which mediatesthe effects of β-chemokines on eosinophils.

The present invention further relates to assays which employ a novelreceptor which is the eosinophil eotaxin receptor. This receptor is ahuman β-chemokine receptor and has been designated “CC CKR3”. One aspectof the present invention is directed to assays employing the humaneosinophil eotaxin receptor, free from receptor-associated proteins. Afurther aspect of this invention is directed to assays which employ thehuman eosinophil eotaxin receptor which is isolated or purified. Inaddition, the present invention provides assays in which the eosinophileotaxin receptor is expressed in an AML14.3D10 cell line.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to an eosinophil eotaxin receptor “CCCKR3” which is a G protein-coupled receptor and has been cloned fromhuman eosinophils and which when stably expressed in AML14.3D10 cellsbinds eotaxin, RANTES and MCP-3 with high affinity. Competition bindingstudies against ¹²⁵I-human eotaxin gives Kd values of 0.1, 2.7, and 3.1nM, respectively for the three β-chemokines. CC CKR3 also binds MCP-1with lower affinity, but does not bind MIP-1α or MIP-1β. Eotaxin,RANTES, and to a lessor extent MCP-3, but not the other chemokinesactivate CC CKR3 as determined by the ability to stimulate a Ca²⁺-fluxin clones expressing the receptor. Competition binding studies onprimary eosinophils give binding affinities for the different chemokineswhich are indistinguishable from those measured with CC CKR3. Since CCCKR3 is prominently expressed in eosinophils it is concluded that CCCKR3 is the eosinophil eotaxin receptor. Eosinophils also express a muchlower level of a second chemokine receptor, CC CKR1, which appears to beresponsible for the effects of MIP-1α.

The eosinophil eotaxin receptor is a protein containing variousfunctional domains, including one or more domains which anchor thereceptor in the cell membrane, and at least one ligand binding domain.As with many receptor proteins, it is possible to modify many of theamino acids, particularly those which are not found in the ligandbinding domain, and still retain at least a percentage of the biologicalactivity of the original receptor. In accordance with this invention, itis suggested that certain portions of the eosinophil eotaxin receptorare not essential for its activation by β-chemokines. Thus thisinvention specifically includes modified functionally equivalenteosinophil eotaxin receptors which have deleted, truncated, or mutatedportions. This invention also specifically includes modifiedfunctionally equivalent eosinophil eotaxin receptors which containmodified and/or deletions in other domains, which are not accompanied bya loss of functional activity.

Additionally, it is possible to modify other functional domains such asthose that interact with second messenger effector systems, by alteringbinding specificity and/or selectivity. Such functionally equivalentmutant receptors are also within the scope of this invention.

A further aspect of this invention are nucleic acids which encode aneosinophil eotaxin receptor or a functional equivalent from human orother species. These nucleic acids may be free from associated nucleicacids, or they may be isolated or purified. For most cloning purposes,cDNA is a preferred nucleic acid, but this invention specificallyincludes other forms of DNA as well as RNAs which encode an eosinophileotaxin receptor or a functional equivalent.

Yet another aspect of this invention relates to vectors which comprisenucleic acids encoding an eosinophil eotaxin receptor or a functionalequivalent. These vectors may be comprised of DNA or RNA; for mostcloning purposes DNA vectors are preferred. Typical vectors includeplasmids, modified viruses, bacteriophage and cosmids, yeast artificialchromosomes and other forms of episomal or integrated DNA that canencode an eosinophil eotaxin receptor. It is well within the skill ofthe ordinary artisan to determine an appropriate vector for a particulargene transfer or other use.

A further aspect of this invention are host cells which are transformedwith a gene which encodes an eosinophil eotaxin receptor or a functionalequivalent. The host cell may or may not naturally express an eosinophileotaxin receptor on the cell membrane. Preferably, once transformed, thehost cells are able to express the eosinophil eotaxin receptor or afunctional equivalent on the cell membrane. Depending on the host cell,it may be desirable to adapt the DNA so that particular codons are usedin order to optimize expression. Such adaptations are known in the art,and these nucleic acids are also included within the scope of thisinvention.

The receptors of this invention were cloned from RNA isolated fromeosinophils. Degenerate PCR was used with primers designed from bothCCCKR1 and CCCKR2, and clones screened by expression in the AML14.3D10cell line. The cloning was made difficult by several factors. First,prior to this invention there was very little information availableabout the biochemical characteristics and intracellularsignalling/effector pathways used by these receptors making screeningprocedures uncertain. Second, this receptor could not be expressedand/or functionally coupled in the cell lines normally used for cloningreceptors such, as COS, CHO, HEK293. After repeated failures usingstandard lines, an obscure eosinophilic-like cell line, AML14.3D10, wastried and found to suitable for expression of the receptors described inthis invention.

The present invention further relates to assays which employ a novelreceptor which is the eosinophil eotaxin receptor. This receptor is ahuman β-chemokine receptor and has been designated “CC CKR3”. One aspectof the present invention is directed to assays employing the humaneosinophil eotaxin receptor, free from receptor-associated proteins. Afurther aspect of this invention is directed to assays which employ thehuman eosinophil eotaxin receptor which is isolated or purified. Inaddition, the present invention provides assays in which the eosinophileotaxin receptor is expressed in an AML14.3D10 cell line.

A particular embodiment of this invention is directed to an assay todetermine the presence of a compound which binds to the eosinophileotaxin receptor. Thus, this invention also comprises a method todetermine the presence of a compound which binds to an eosinophileotaxin receptor comprising:

(a) introducing a nucleic acid which encodes an eosinophil eotaxinreceptor into a cell under conditions so that eosinophil eotaxinreceptor is expressed;

(b) introducing a detector molecule or a nucleic acid encoding adetector molecule into the cell, wherein the detector molecule isdirectly or indirectly responsive to a eosinophil eotaxin-ligand bindingevent;

(c) contacting the cell with a compound suspected of binding to theeosinophil eotaxin receptor; and

(d) determining whether the compound binds to the eosinophil eotaxinreceptor by monitoring the detector molecule.

In a preferred embodiment of the present invention, the eosinophileotaxin receptor is expressed in AML14.3D10 cells.

In another preferred embodiment of the present invention, the binding ofthe compound suspected of binding to the eosinophil eotaxin receptor iscompared to the binding or the influence of eotaxin, RANTES and MCP-3.

A further embodiment of this invention is directed to an assay todetermine the presence of a compound which antagonizes the binding of aknown ligand to the eosinophil eotaxin receptor. Thus, this inventionfurther comprises a method to determine the presence of a compound whichantagonizes the eosinophil eotaxin receptor comprising:

(a) introducing a nucleic acid which encodes the eosinophil eotaxinreceptor into a cell under conditions so that eosinophil eotaxinreceptor is expressed;

(b) introducing a detector molecule or a nucleic acid encoding adetector molecule into the cell, wherein the detector molecule isdirectly or indirectly responsive to an eosinophil eotaxin-ligandantagonism event;

(c) contacting the cell with a compound suspected of antagonizing theeosinophil eotaxin receptor;

(d) contacting the cell with a compound which is a known ligand of theeosinophil eotaxin receptor; and

(e) determining whether the compound antagonizes the action of the knownligand to the eosinophil eotaxin receptor by monitoring the detectormolecule.

In a preferred embodiment of the present invention, the eosinophileotaxin receptor is expressed in AML14.3D10 cells.

In another preferred embodiment of the present invention, the knownligand of the eosinophil eotaxin receptor is eotaxin, RANTES and MCP-3.

One aspect of this invention is the development of a sensitive, robust,reliable and high-throughput screening assay which may be used to detectligands which bind to the eosinophil eotaxin receptor, in particular,antagonists of the action of chemokines on eosinophils.

In particular, a typical protocol of such an assay is as follows. Assaybuffer (50 mM Hepes, pH 7.2 w/0.5% BSA, 5 mM MgCl₂, 1 mM CaCl₂, 100 uMPMSF and 10 ug/ml phosphoramidon, leupeptin, aprotinin and chymostatin),test compound (or equivalent volume of solvent), 20 pM ¹²⁵I-humaneotaxin (2000 Ci/mmol), 25 ng unlabeled human eotaxin (non-specificbinding wells only), and AML14.3D10 cells expressing eotaxin receptorcells, or eosinophils, are added sequentially in 96-well, round-bottom,polystyrene plates to a final volume of 250 uL. Assay plates are thenmixed and incubated for 60 minutes at 31° C. After incubation, assayplates are harvested onto Packard 96-well GF/C Unifilter plates treatedwith 0.33% polyethylenimine (PEI) using Packard Filtermate 196 cellharvester. Wells and filters are washed with 200 uL 50 mM Hepes, pH 7.2with 0.5M NaCl and 0.02% NaN₃. After filtration, GF/C plates are driedand sealed. 25 uL Packard Microscint-O scintillant are then added toeach well and counted for 2 minutes on Packard Topcount (liquid ¹²⁵ Isetting).

Ligands detected using assays described herein may be used in thetreatment and prevention of conditions which would be benefited by themodification of the activity of the eosinophil eotaxin receptor, such asin the treatment and prevention of atopic conditions including allergicrhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma.

A further aspect of this invention is directed to novel ligands whichare identified using the subject assays.

The eosinophil eotaxin receptor and fragments are immunogenic. Thus,another aspect of this invention is antibodies and antibody fragmentswhich can bind to eosinophil eotaxin receptor or an eosinophil eotaxinreceptor fragment. These antibodies may be monoclonal antibodies andproduced using either hybridoma technology or recombinant methods. Theymay be used as part of assay systems or to deduce the function of aneosinophil eotaxin receptor present in a cell.

A further aspect of this invention are antisense oligonucleotidesnucleotides which can bind to eosinophil eotaxin receptor nucleotidesand modulate receptor function or expression.

A further aspect of this invention is a method of increasing the amountof eosinophil eotaxin receptor in a cell comprising, introducing intothe cell a nucleic acid encoding an eosinophil eotaxin receptor, andallowing expression of the eosinophil eotaxin receptor.

As used throughout the specification and claims, the followingdefinitions shall apply:

Ligand—any molecule which binds to an eosinophil eotaxin receptor ofthis invention. These ligands can have either agonist, partial agonist,partial antagonist or antagonist activity.

Free from receptor-associated proteins—the receptor protein is not in amixture or solution with other membrane receptor proteins.

Free from associated nucleic acids—the nucleic acid is not covalentlylinked to DNA which it is naturally covalently linked in the organism'schromosome.

Isolated receptor—the protein is not in a mixture or solution with anyother proteins.

Isolated nucleic acid—the nucleic acid is not in a mixture or solutionwith any other nucleic acid.

Functional equivalent—a receptor which does not have the exact sameamino acid sequence of a naturally occurring eosinophil eotaxinreceptor, due to alternative splicing, deletions, mutations, oradditions, but retains at least 1%, preferably 10%, and more preferably25% of the biological activity of the naturally occurring receptor. Suchderivatives will have a significant homology with the natural eosinophileotaxin receptor and can be detected by reduced stringency hybridizationwith a DNA sequence obtained from an eosinophil eotaxin receptor. Thenucleic acid encoding a functional equivalent has at least about 50%homology at the nucleotide level to a naturally occurring receptornucleic acid.

Purified receptor—the receptor is at least about 95% pure. Purifiednucleic acid—the nucleic acid is at least about 95% pure.

Single-letter abbreviations for amino acid residues are as follows: A,Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L,Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W,Trp; and Y, Tyr.

Orphan Cloning of an Eosinophil Chemokine Receptor

RT/PCR conducted using oligonucleotide primers developed from the aminoacid residues clustered within transmembrane helicies II (TMII) and VII(TMVII) of the β-chemokine receptors, CC CKR1 (Neote, K., et al. (1993)Cell 72, 415-425) and MCP-1R (Charo, I. F., et al. (1994) Proceecing ofthe National Academy of Sciences 91, 2752-2756) on total RNA isolatedfrom eosinophils yielded DNA fragments of ˜700 bases, a size consistentwith that expected for a G protein coupled receptor. Analysis of severalTMII to TMVII clones provided a novel sequence which was 76% homologouswith human CC CKR1 at the nucleic acid level. Completion of the cloningof the 3′ and 5′ ends gave a sequence for a protein of 355 residues inlength, 63% identical to CC CKR1, and 51% identical to CC CKR2B, itsclosest homologues.

The amino acid sequence of the human eosinophil eotaxin receptor CC CKR3is depicted below (SEQ ID NO:1): Met Thr Thr Ser Leu Asp Thr Val Glu ThrPhe Gly Thr Thr Ser Tyr Tyr Asp Asp Val Gly Leu Leu Cys Glu Lys Ala AspThr Arg Ala Leu Met Ala Gln Phe Val Pro Pro Leu Tyr Ser Leu Val Phe ThrVal Gly Leu Leu Gly Asn Val Val Val Val Met Ile Leu Ile Lys Tyr Arg ArgLeu Arg Ile Met Thr Asn Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu LeuPhe Leu Val Thr Leu Pro Phe Trp Ile His Tyr Val Arg Gly His Asn Trp ValPhe Gly His Gly Met Cys Lys Leu Leu Ser Gly Phe Tyr His Thr Gly Leu TyrSer Glu Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu Ala Ile ValHis Ala Val Phe Ala Leu Arg Ala Arg Thr Val Thr Phe Gly Val Ile Thr SerIle Val Thr Trp Gly Leu Ala Val Leu Ala Ala Leu Pro Glu Phe Ile Phe TyrGlu Thr Glu Glu Leu Phe Glu Glu Thr Leu Cys Ser Ala Leu Tyr Pro Glu AspThr Val Tyr Ser Trp Arg His Phe His Thr Leu Arg Met Thr Ile Phe Cys LeuVal Leu Pro Leu Leu Val Met Ala Ile Cys Tyr Thr Gly Ile Ile Lys Thr LeuLeu Arg Cys Pro Ser Lys Lys Lys Tyr Lys Ala Ile Arg Leu Ile Phe Val IleMet Ala Val Phe Phe Ile Phe Trp Thr Pro Tyr Asn Val Ala Ile Leu Leu SerSer Tyr Gln Ser Ile Leu Phe Gly Asn Asp Cys Glu Arg Ser Lys His Leu AspLeu Val Met Leu Val Thr Glu Val Ile Ala Tyr Ser His Cys Cys Met Asn ProVal Ile Tyr Ala Phe Val Gly Glu Arg Phe Arg Lys Tyr Leu Arg His Phe PheHis Arg His Leu Leu Met His Leu Gly Arg Tyr Ile Pro Phe Leu Pro Ser GluLys Leu Glu Arg Thr Ser Ser Val Ser Pro Ser Thr Ala Glu Pro Glu Leu SerIle Val Phe

The sequence for the cDNA encoding the human eosinophil eotaxin receptorCC CKR3 beginning with nucleotide 3587 and ending with nucleotide 4651is depicted below (SEQ ID NO:2):                                       ATGA CAACCTCACT 3601 AGATACAGTTGAGACCTTTG GTACCACATC CTACTATGAT GACGTGGGCC 3651 TGCTCTGTGA AAAAGCTGATACCAGAGCAC TGATGGCCCA GTTTGTGCCC 3701 CCGCTGTACT CCCTGGTGTT CACTGTGGGCCTCTTGGGCA ATGTGGTGGT 3751 GGTGATGATC CTCATAAAAT ACAGGAGGCT CCGAATTATGACCAACATCT 3801 ACCTGCTCAA CCTGGCCATT TCGGACCTGC TCTTCCTCGT CACCCTTCCA3851 TTCTGGATCC ACTATGTCAG GGGGCATAAC TGGGTTTTTG GCCATGGCAT 3901GTGTAAGCTC CTCTCAGGGT TTTATCACAC AGGCTTGTAC AGCGAGATCT 3951 TTTTCATAATCCTGCTGACA ATCGACAGGT ACCTGGCCAT TGTCCATGCT 4001 GTGTTTGCCC TTCGAGCCCGGACTGTCACT TTTGGTGTCA TCACCAGCAT 4051 CGTCACCTGG GGCCTGGCAG TGCTAGCAGCTCTTCCTGAA TTTATCTTCT 4101 ATGAGACTGA AGAGTTGTTT GAAGAGACTC TTTGCAGTGCTCTTTACCCA 4151 GAGGATACAG TATATAGCTG GAGGCATTTC CACACTCTGA GAATGACCAT4201 CTTCTGTCTC GTTCTCCCTC TGCTCGTTAT GGCCATCTGC TACACAGGAA 4251TCATCAAAAC GCTGCTGAGG TGCCCCAGTA AAkAAAAGTA CAAGGCCATC 4301 CGGCTCATTTTTGTCATCAT GGCGGTGTTT TTCATTTTCT GGACACCCTA 4351 CAATGTGGCT ATCCTTCTCTCTTCCTATCA ATCCATCTTA TTTGGAAATG 4401 ACTGTGAGCG GAGCAAGCAT CTGGACCTGGTCATGCTGGT GACAGAGGTG 4451 ATCGCCTACT CCCACTGCTG CATGAACCCG GTGATCTACGCCTTTGTTGG 4501 AGAGAGGTTC CGGAAGTACC TGCGCCACTT CTTCCACAGG CACTTGCTCA4551 TGCACCTGGG CAGATACATC CCATTCCTTC CTAGTGAGAA GCTGGAAAGA 4601ACCAGCTCTG TCTCTCCATC CACAGCACAG CCGGAACTCT CTATTGTGTT 4651 Tor a degenerate variation thereof.

The 5′ genomic DNA flanking sequence encoding the human eosinophileotaxin receptor further comprises the region beginning with nucleotide1 and ending with nucleotide 3586 as depicted below (SEQ ID NO:3):    1GGATCCCTAC CTTCCCCATC AGAGCTAGGG GGCATGGAGC GCTCTCTGCT   51 AAGATGGGGACCCCCAAGGA ATGTCTCCCT GTGGGGCACT TCCTTACCAG  101 ATGGGATGGC CAGTGCGGTTAAGTTGGTGG TCAGGCAGAA AAAAAAGATC  151 TAGTTTGTAC TCTTGAGAGT TCCTCGGTTTGTTCATGGCA TGGGCAGGGA  201 GTCAAGGAGC AGCAGCCTTG CCTCAGTGCC TACCAGTGCAGGAAAAGGTG  251 CATAGCCTGG GCCAGGGCCA GGGCCCTGGT GGAGGCGTAG TGGTAACAGA 301 GAGGGCTCTC CATTCCAGCC CAAGGAAGAC TAAGAATGAA TACCTCATGA  351CTATATTAGC TACAAACCAC CACAGCAGGT TCCAGAAAAA GGCTCAGCGT  401 TGGAACCAGGTCACCCCCAC TCAGCAGACA CCAGTCATAT AAATCAAGGA  451 CCAACAGGAG ACAGGAACACCCCCTTCCCA CTCTGCCCCA TGTCTCAAGT  501 TGTAGTGGCC CTTCCTCcAG ATCTCTGCCACCATCTTAGA AAGGAACACT  551 GAAAGAAGAA ACTGAAATTA TAAGCTGACA GCATAAAGAGGATGAGTAAA  601 ACCTAAAATC ATTGTTCACA TGAATGAATC AAGAGAAGTT TAAACCACTT 651 TGGACTAAAA TGTGTGAATC CTTTTTCCTG CTATCCAGCA GATGAGAAGC  701TGGTAACAGA GACCACAATA GTTTGGAGAC TAAAGAATCA TTGCACATTT  751 CACTGCTGAGTTGTATTGTG AGTAATTTTA GTTGACCTCA CTTTGTAAAT  801 CTTGCACACG GGGCAATCCAATATCTGCAC AAGAGATATG TTAACCAGTG  851 GTAAATGCTG CATGAGGAGA TTGGGTGATTTTTACTTTCG TTTTTGTGCT  901 CTTCTTTCTT ATTGTTCTTA CTTATTTACG ATTACCCTATCGTTTTCCCA  951 AAATGTAAAA GGCCATTTTG AAAGCCTAAT TCAAACCTCT TCACTATTTT1001 GTATCTAAGT ATTCACCTTG ATTGAGACTG GGTAGACAGG TGAAAACCAT 1051ATCAGGTTTT TAATTTTTTA ATTTTTAATT ATTTATTTAT TTATTTATTT 1101 TTTGAGATGGAGTCTGGCTG TCGCCCAGGC TGGAGTGCAG CGGCGTGATC 1151 ACAGTTCACT GCAGCCTCAACCTTCTAGGC TCAAGGGATT CTCCCACCTC 2601 GAAATCCCAT TGACTGACCC CTCCTGCTTACCCCTTTGTG ATGGAGAAGC 2651 TCCCAGGGGT TTGCTTTTTG CATGTTACCA GGCCTAACTCAGCATCACCA 2701 GGGGCAAGAA AAGGAAAGTA ACCTAAACTA ATGCTGCTTA TAATTGTAAT2751 TATTGTAATA GTTAATTACT GTGATTGTAC ATGTGTAACA GACAAAATGT 2801GTATTTTTTT CACAGCTGCT GTGGATTGGA TTATGCCATT TGGAATAAGA 2851 ATGCTGTTAAGAGCACACAA GCCAGGTTCC TCAAGTCCGT AGCAAATTTT 2901 TCAAAAGTTA AATTTAAAAATCACTACATT TGAATCTAGT GACAGGAGAA 2951 ATGGACATGG ATAGAGACTA AAGATCTAGCCCAAATTTTA TATTTACTTG 3001 TTAGAGGATT TTGAACAAAT TACTAAATTT CTTCAAGGTTCAATTTCCCC 3051 ATTAACTATA ATGAATGTCT CATCATTATG GGGCCCTGGA GAAGCATAAT3101 TACTTGTAAT TGTAATAATC ATTGTTATTA TTATTATACA TATTTTGCTT 3151TTAAATGGAT AAGGATTTTT AAGGTATATG TAAACTGTAA AACATAAPAT 3201 GCAAAATGCCGTAAGAGACA GTAGTAATAA TAATGATTAT TATATTGTTA 3251 TCATTATCTA GCCTGTTTTTTCCTGTTGTG TATTTCTTCC TTTAAATGCT 3301 TACAGAAATC TGTATCCCCA TTCTTCACCACCACCCCACA ACATTTCTGC 3351 TTCTTTTCCC ATGCCGGTCA TGCTAACTTT GAAAGCTTCAGCTCTTTCCT 3401 TCCTCAATCC TTCTCCTGGC ACCTCTGATA TGCCTTTTGA AATTCATGTT3451 AAAGAATCCC TAGGCTGCTA TCACATGTGG CATCTTTGTT GAGTACATGA 3501ATAAATCAAC TGGTGTGTTT TACGAAGGAT GATTATGCTT CATTGTGGGA 3551 TTGTATTTTTCTTCTTCTAT CACAGGGAGA AGTGAAor a degenerate variation thereof.

The sequence for the cDNA encoding human eosinophil eotaxin receptorfurther comprises the terminator region beginning with nucleotide 4652and ending with nucleotide 5099 as depicted below (SEQ ID NO:4): 4652TAGGTCAGA TGCAGAAAAT TGCCTAAAGA GGAAGGACCA AGGAGATGAA 4701 GCAAACACATTAAGCCTTCC ACACTCACCT CTAAAACAGT CCTTCAAACT 4751 TCCAGTGCAA CACTGAAGCTCTTGAAGACA CTGAAATATA CACACACCAG 4801 TAGCAGTAGA TGCATGTACC CTAAGGTCATTACCACAGGC CAGGGGCTGG 4851 GCAGCGTACT CATCATCAAC CCTAAAAAGC AGAGCTTTGCTTCTCTCTCT 4901 AAAATGAGTT ACCTACATTT TAATGCACCT GAATGTTAGA TAGTTACTAT4951 ATGCCGCTAC AAAAAGGTAA AACTTTTTAT ATTTTATACA TTAACTTCAG 5001CCAGCTATTG ATATAAATAA AACATTTTCA CACAATACAA TAAGTTAACT 5051 ATTTTATTTTCTAATGTGCC TAGTTCTTTC CCTGCTTAAT GAAAAGCTTor a degenerate variation thereof.

As will be appreciated by one skilled in the art, there is a substantialamount of redundancy in the set of codons which translate specific aminoacids. Accordingly, this invention also includes alternative basesequences wherein a codon (or codons) are replaced with another codonsuch that the amino acid sequence translated by the DNA sequencesremains unchanged. For purposes of this specification, a sequencebearing one or more such replaced codons will be defined as a degeneratevariation. Also included are mutations (exchanges of individual aminoacids) which one skilled in the art would expect to have no effect onfunctionality, such as valine for leucine, arginine for lysine, andasparagine for glutamine.

The amino acid sequence of CC CKR3 shares some sequence homology with CCCKR1 (Neote, K., et al. (1993) Cell 72, 415-425), CC CKR2B (Charo, I.F., et al. (1994) Proceecing of the National Academy of Sciences 91,2752-2756), CC CKR4 (Power, C. A., et al. (1995) Journal of BiologicalChemistry 270, 19495-19500) and V28 (Raport, C. J., et al. (1995) Gene163, 295-299). The sequence of this protein, designated CC CKR3, iscomparable to that previously reported by Combadiere et al. (Combadiere,C., et al. (1995) Journal of Biological Chemistry 270, 16491-16494)except that it contains a lysine in place of asparagine at position 107.Genomic cloning provided confirmation of the subject sequence, includinglysine at position 107. The sequence discrepancy, which results from asubstitution of G to T at the third position of the codon for residue107, could represent a genetic polymorphism. This is highly unlikely,however, because all α- and β-chemokine receptors analyzed to datecontain lysine in that position including the recently describedbasophilic β-chemokine receptor (Power, C. A., et al. (1995) Journal ofBiological Chemistry 270, 19495-19500), CC CKR1 (Neote, K., et al.(1993) Cell 72, 415-425), MCP-1R (Charo, I. F., et al. (1994) Proceecingof the National Academy of Sciences 91, 2752-2756), IL-8RA and IL-8RB(Holmes, W. E., et al. (1991) Science 253, 1278-1280; Murphy, P. M., etal. (1991) Science 253, 1280-1283), the three murine β-chemokinereceptors (Post, T. W., et al. (1995) Journal of Immunology 155,5299-5305; Gao, J. L., et al. (1995) Journal of Biological Chemistry270, 17494-17501) as well as three human chemokine-like receptors(Loetscher, M., et al. (1994) Journal of Biological Chemistry 269,232-237; Raport, C. J., et al. (1995) Gene 163, 295-299; Combadiere, C.,et al. (1995) DNA and Cell Biology 14, 673-680; Federsppiel, B., et al.(1993) Genomics 16, 707-712). An unusual feature of CC CKR3 is thecluster of negatively charged amino acids (ETEELFEET) distal to TMIV inthe second extracellular loop.

Expression of the Human CC CKR3 in AML14.3D10 Cells

Once a full length cDNA encoding CC CKR3 was isolated and cloned intothe expression vector pBJ/NEO the resulting plasmid designatedpBJ/NEO/CCCKR3, was transfected into the AML14.3D10 line.

The CC CKR3 transfected AML14.3D10 cell line has been placed onrestricted deposit with American Type Culture Collection in Rockville,Md. as ATCC No. CRL-12079, on Apr. 5, 1996.

Stable clones were selected for neomycin resistance, and a number werechosen for further analysis. To demonstrate expression of receptorprotein, a western blot was performed using antisera generated against apeptide derived from the predicted C-terminus of CC CKR3. Immunoreactivebands migrating at approximately 45-55 kd are present in primaryeosinophils and the 3.16 clone, indicating that CC CKR3 is indeedexpressed in these cells. There was no immunoreactive bands present inneutrophils indicating that the antisera was indeed identifying aneosinophil-specific protein. A nonspecific pattern of immuno-reactivitywas detected in untransfected AML14.3D10 cells, and furthermore, thispattern was identical in clone 3.49 indicating that thisneomycin-resistant clone is a non-expressor of CC CKR3. Of the 27neomycin resistant clones studied, 19 failed to express CC CKR3. Theother 8 did express the receptor as judged both by Western analysis, andby the ability of eotaxin and RANTES to induce Ca²⁺-fluxes. Thenon-expressing clones were used as negative controls in subsequentexperiments.

Binding to CC CKR3 on Intact AML14/CCCKR3.16 Cells

Because preliminary experiments with three different CC CKR3 expressingclones indicated that they bound ¹²⁵I-eotaxin, competition studies usingthis labeled ligand were performed to characterize the bindingproperties of the receptor. As shown in Table 1, unlabeled human eotaxincompeted with an Kd of 0.1 nM. Results with murine eotaxin wereessentially identical. Scatchard analysis demonstrated that eotaxinbinds with a single affinity and that the different clones expressed2−4×10⁵ receptors/cell. The ability to bind eotaxin is due to CC CKR3since neither immunoreactive negative clones, such as 3.49, noruntransfected cells displayed any specific binding. Clearly, CC CKR3 isa high affinity receptor for eotaxin. Cross-competition studies with thetwo other β-chemokines known to be eosinophil chemoattractants, RANTESand MCP-3, demonstrated that they too have considerable affinity for CCCKR3, with Kd's of about 3 nM (See Table 1). In contrast, MCP-1 competedwith much lower affinity (Kd=60 nM), and MIP-1α, and MIP-1β failed tocompete at all (See Table 1). Similarly, the α-chemokine IL-8 did notinhibit eotaxin binding.

Competition studies were also carried out against ¹²⁵I-MCP-3. Again,human and murine eotaxin competed strongly with Kd's of 0.2 and 0.3 nM(Table 1). RANTES and MCP-3 also demonstrated high affinity with Kd's of0.5 and 0.7 nM, values about 4-fold lower than observed against eotaxin.As in the studies with eotaxin, MCP-1 competed weakly (Kd=16 nM), andMIP-1α, and MIP-1β failed to compete at all. Thus despite some smallquantitative differences the overall ligand selectivity of the receptoris the same whether measured by competition against eotaxin or MCP-3,and the order of potency, eotaxin>MCP-3=RANTES>>MCP-1, is identical.

CC CKR3 is Functionally Coupled in AML14.3D10 Cells

In order to determine whether human CC CKR3 was functionally coupledwhen expressed in the AML14.3D10 line, intracellular Ca²⁺ levels weremeasured in response to various 3-chemokines. Both 100 nM eotaxin andRANTES induced Ca²⁺-fluxes in cells expressing the receptor.Surprisingly, 1 μM of MCP-3 was required to induce a response, and thatresponse was smaller than those observed for eotaxin or RANTES. Noresponse at all was generated by addition of MIP-1α, MIP-1β, MCP-1 orIL-8 at concentrations as high as 1 μM. The responses to eotaxin,RANTES, and MCP-3 are due to the specific expression of CC CKR3 sincenone of these mediators induced fluxes in untransfected cells or inclone 3.49. While the preliminary functional characterization byCombadiere et. al. differs greatly from the present invention, they werenot able to demonstrate any specific binding to cells putativelyexpressing the receptor, and such functional data have now beenretracted (Combadiere, C., et al. (1995) Journal of Biological Chemistry270, 30235).

Binding Properties of Primary Eosinophils

The selectivity of CC CKR3 for the various β-chemokines mirrors theeffectiveness of these ligands as eosinophil chemoattractants suggestingthat CC CKR3 is the primary mediator of chemokine induced eosinophilchemotaxis. To provide additional pharmacological evidence, bindingstudies were conducted on primary eosinophils. When measured bycompetition against ¹²⁵I-eotaxin, unlabeled human eotaxin gave an Kd=0.1nM, a value identical to that obtained on cloned CC CKR3 (see Table 1).Scatchard showed a single binding affinity, and 4×10⁵ sites/cell. Thenumber of binding sites varied by less than 2-fold for the 3 donors usedin the studies. The affinites for RANTES and MCP-3 were also identicalto those measured on CC CKR3, and as with CC CKR3, neither MIP-1α, orMIP-1β, showed any ability to compete with radiolabeled eotaxin (seeTable 1). Similarly, the Kd's obtained by competition against ¹²⁵I-MCP-3on eosinophils are effectively indistinguishable to those measuredagainst cloned CC CKR3 (see Table 1). All of the observations andmeasurements, taken together with the Western blot showing expression ofCC CKR3, verify that CC CKR3 is the eosinophil eotaxin receptor, andappears to be largely responsible for mediating the effects of mostβ-chemokines on eosinophils.

Stably expressed in the eosinophilic line AML14.3D10, CC CKR3 bindseotaxin, RANTES and MCP-3, with high affinity, with a rank order ofpotency of eotaxin>RANTES=MCP-3. MCP-1 binds with much lower affinity,while MIP-1α and MIP-1β fail to bind at all. The selectivity of CC CKR3mirrors most of the binding activity of primary eosinophils. In fact,when measured by competition against 1251-eotaxin, the bindingaffinities on eosinophils for all of these β-chemokines areindistinguishable from those obtained with cloned CC CKR3. Moreover, CCCKR3 was cloned from eosinophils, and as shown by Western blotting isheavily expressed in these cells. The abilities of the differentchemokines to activate CC CKR3 are consistent with the binding data aseotaxin, RANTES, and to a lessor extent MCP-3 all stimulate Ca²⁺ fluxesin clones which express the receptor, while MCP-1, MIP-1α and MIP-1β donot, even at concentrations as high as 1 μM. Thus, based on itsproperties, and expression, CC CKR3, is the eosinophil eotaxin receptor.TABLE 1 Binding affinities of various chemokines comparing CC CKR3expressed in AML14.3D10 with primary eosinophils K_(d) (nM) competitorCC CKR3 eosinophils ¹²⁵I-human eotaxin human-eotaxin 0.1 ± 0.04 (4) 0.1± 0.03 (3) murine-eotaxin 0.1 ± 0.04 (3) 0.1 ± 0.01 (2) MCP-3 2.7 ± 1.7(5) 3.0 ± 0.2 (2) RANTES 3.1 ± 0.6 (5) 2.6 ± 0.3 (2) MCP-1  60 ± 9 (3) 41 ± 2 (2) MIP-1a N.B. (4) N.B. (2) MIP-1β N.B. (4) N.B. (2) ¹²⁵I-MCP-3human-eotaxin 0.2 ± 0.1 (4) 0.2 ± 0.1 (2) murine-eotaxin 0.3 ± 0.1 (2)0.2 ± 0.1 (3) MCP-3 0.7 ± 0.4 (4) 1.1 ± 0.6 (10) RANTES 0.5 ± 0.3 (4)0.9 ± 0.4 (8) MCP-1  16 ± 2 (3)  61 ± 13 (2) MIP-1a N.B. (4) see textMIP-1β N.B. (4) N.B. (2)

Competition binding experiments were carried out against the indicatediodinated ligand as follows and as described herein. Equilibrium bindingof β-chemokines to AML14.3D10 cells expressing CC CKR3 and to primaryeosinophils was examined with increasing concentrations of unlabelledhuman eotaxin, murine eotaxin, RANTES, MCP-3, or MCP-1 to competeagainst fixed concentrations of either ¹²⁵I-human eotaxin, or¹²⁵I-MCP-3. Also the competition with 100 nM concentrations of MIP-1α,and MIP-1β was examined. The experiments were carried out either withAML14.3D10 cells expressing CC CKR3, or with eosinophils. All values arethe averages of triplicate determinations. Typically, 4000-6000 cpm ofiodinated ligand was bound in the absence of compeititor with S/N ratiosexceeding 15. Human and murine eotaxin are the human and murinechemokines, respectively. “N.B.” means that no competition was observed.All results are the averages of the number of experiments shown inparenthesis.

Various changes and modifications may be made in the products andprocesses of the present invention without departing from the spirit andscope thereof. The various embodiments and the examples which have beenset forth herein are given for the purpose of illustrating the presentinvention and shall not be construed as being limitations on the scopeor spirit of the instant invention.

EXAMPLE 1

mRNA Isolation and cDNA Cloning

Total RNA was isolated from purified eosinophils with TRIzol reagent(BRL) and used in a RT/PCR reaction (Daugherty, B. L., et al. (1991)Nucleic Acids Research 19, 2471-2476) using oligonucleotide primersdesigned from the human CC CKR1 and MCP-1RB cDNA sequences (Neote, K.,et al. (1993) Cell 72, 415-425; Charo, I. F., et al. (1994) Proceecingof the National Academy of Sciences 91, 2752-2756). The primers used forPCR corresponded to a consensus sequence encoded in transmembranedomains (TM) II and VII:

5′-PCR primer (TMII) (SEQ ID NO:5): 5′-AACCTGGCCAT(C,T)TCTGA(C,T)CTGC-3′

3′-RT/PCR primer (TMVII) (SEQ ID NO:6):5′-GAAC(C,T)TCTC(C,A)CCAACGAAGGC.

The resultant PCR product of ˜700 bp was subcloned into plasmid pNoTA(Five Prime, Three Prime, Inc) and sequenced using Sequenase (USB). Theremaining 5′ and 3′ sequence encoding CC CKR3 was cloned by rapidamplification of cDNA ends (RACE) using both the 5′-RACE and 3′-RACEkits (Clontech) with the following primer sequences:

(5′-RACE) (SEQ ID NO:7): 5′-TCTCGCTGTACAAGCCTGTGTG-3′;

(3′-RACE) (SEQ ID NO:8): 5′-CCTTCTCTCTTCCTATCAATCC-3′.

The resultant PCR products (5′-RACE, ˜450 bp; 3′-RACE, ˜700 bp) weresubcloned into pCRII (Invitrogen) and sequenced. Upon identification ofthe 5′-end of the cDNA containing the initiator ATG codon and the 3′-endcontaining the termination codon TAG, a new set of PCR primers weredesigned to reamplify the entire coding region from eosinophil total RNAfor expression of CC CKR3. The primer sequences used for RT/PCR were:

5′-PCR primer (SEQ ID NO:9):5′-ATATATTAAGCTTCCACCATGACAACCTCACTAGATACAG-3′;

3′-RT/PCR primer (SEQ ID NO:10):5′-ATATATTCTAGAGCGGCCGCTAAAACACAATAGAGAGTTCC-3′.

The resultant PCR product of 1105 bp was digested with HindIII and NotIand subcloned into plasmid pBJ/NEO to yield pBJ/NEO/CCCKR3. The plasmidpBJ/NEO was prepared essentially as follows. Plasmid pD5/Igh/Neo(Daugherty, B. (1991) Nucleic Acids Research 19, 2471-2476) was digestedwith the restriction enzyme SalI, filled in with E. coli DNA polymeraseI Kienow fragment to create a blunt end and subsequently digested withthe restriction enzyme NotI. The CMVIE intron A fragment from plasmid p89-1 was digested with ClaI, filled in to create a blunt end andsubsequently digested with HindIII. These fragments were used in athree-way ligation with a HindIII and NotI fragment of the human C5areceptor cDNA. The C5a receptor fragment was excised with HindIII andNotI and replaced with the eotaxin receptor cDNA of 1105 bp obtained byRT/PCR with oligonucleotides SEQ ID NO:9 and SEQ ID NO: 10 afterdigestion with HindIII and NotI. Several clones were sequenced and oneclone comprising the consensus sequence was chosen for expression of CCCKR3 in heterologous cells.

EXAMPLE 2

Transfection into AML14.3D10 Human Eosinophilic Cell Line

AML14.3D10 cells (Paul, C. C., et al. (1995) Blood 86, 3737-3744) werecultured in RPMI-1640, 10% FBS, 1 mM sodium pyruvate, 0.5 μMβ-mercaptoethanol and 2 mM L-glutamine (complete medium). Cells wereharvested at a density of 0.3×10⁶/mL, washed once in PBS, resuspended inRPMI at 10⁷/mL, and 25 μg of plasmid was added. Electroporation wascarried out at 300 V, 960 μF using a Gene Pulser (BioRad). Followingelectroporation, cells were chilled at 0° C. for 10 min and then platedin complete medium at 106/T75 flask and cultured at 37° C., 5% CO₂.After 16-24 hr, cells were pelleted and resuspended in complete mediumcontaining 2 mg/mL Geneticin (GIBCO). Cells were maintained in selectionmedium for 8-10 days until individual surviving clusters appeared.Individual cells were then transferred to 96-well plates and expanded.AML14/CCCKR3 sublines were assayed for the ability to generate a Ca²⁺flux in response to either RANTES or eotaxin. Positive sublines werethen probed by western blotting with an antibody raised against thepredicted C-terminus of CC CKR3. Cell lines positive in both sets ofassays were then characterized for their ability to bind to a variety ofCC chemokines, including eotaxin, RANTES, MCP-3, MIP-1α, MIP-1β andMIP-1.

EXAMPLE 3

Purification of Eosinophils

Primary eosinophils were isolated from granulophoresis preparationsobtained from allergic and asthmatic donors (Bach, M. K., et al. (1990)Journal of Immunological Methods 130, 277-281). The leukocytes weremixed with equal volumes of HBSS and layered over LSM (Organon Teknika)as described (Rollins, T. E., et al. (1988) Journal of BiologicalChemistry 263, 520-526). After lysis of erythrocytes with NH₄Cl, thegranulocytes were subsequently treated with anti-CD 16 microbeadsfollowed by MACS separation (Miltenyl Biotech) (Hansel, T. T., et al.(1991) Journal of Immunological Methods 145, 105-110). Typically theeosinophil preparations were >99% pure as determined using the LeukoStatstaining kit (Fisher).

EXAMPLE 4

Generation of α-CC CKR3 Antisera and Immunoblotting

Polyclonal rabbit antisera was generated to CC CKR3 using the C-terminaldecapeptide sequence TAEPELSIVF. Peptide synthesis, coupling tothyroglobulin and production of antisera was performed (Miller, D. K.,et al. (1993) Journal of Biological Chemistry 268, 18062-18069). Wholecells were boiled and sonicated in Laemli sample buffer (Laemmli, U. K.(1970) Nature 227, 680-685), electrophoresed on 4-20% SDS gels (Novex),transferred to polyvinylidene difluoride membranes (BioRad), and blockedwith 5% nonfat dry milk in TBST (20 mM Tris, 200 mM NaCl, 0.1% Tween-20)for 16 hr at 4° C. The membrane was incubated with antisera at 1:1000 inTBST for 1 hr at room temperature, washed, and subsequently incubatedwith goat anti-rabbit HRP (Zymed) at 1:4000 in TBST for 30 min also atroom temperature. After washing, the membrane was treated with ECLwestern blotting reagents (Amersham) for 1 min, covered in plastic wrapand exposed to film for 2 min.

EXAMPLE 5

Chemokine Binding Assays

Recombinant MCP-3, MCP-1, RANTES, murine and human eotaxin were obtainedfrom Peprotech (Princeton, N.J.). ¹²⁵I-MCP-3 and ¹²⁵I-MIP-1α wereobtained from New England Nuclear (Boston, Mass.), and¹²⁵I-human-eotaxin was obtained from Amersham. Binding of ¹²⁵I-labeledligands (typically a total of 2×10⁴ cpm) in the presence of varyingconcentrations of unlabeled ligands to intact cells (typically 1.5×10⁴,10⁵, or 10⁶ for experiments with labeled eotaxin, MCP-3, or MIP-1α,respectively) were performed at 32° C. (Van Riper, G., et al. (1993)Journal of Experimental Medicine 177, 851-856).

EXAMPLE 6

Ligand-Induced Ca²⁺ Fluxes

Human CC CKR3 expressing AML14 clones or purified eosinophils wereincubated with 1.25 μg/ml Indo-I (Molecular Probes, Eugene, Oreg.) inRPMI 1640, 10 mM HEPES, 5% FBS, for 60 min at 37° C. (Van Riper, G., etal. (1993) Journal of Experimantal Medicine 177, 851-856). Loaded cellswere washed and incubated at 37° C. before the addition of ligands.Calcium fluxes were performed on a FACS analyzer (Becton Dickinson &Co., Mountain View, Calif.) with an excitation wavelenth of 365 nm anddual emission wavelength of 405 and 488 nm.

EXAMPLE 7

CC CKR3 Binding Assay

Assay buffer (50 mM Hepes, pH 7.2 w/0.5% BSA, 5 mM MgCl₂, 1 mM CaCl₂,100 uM PMSF and 10 ug/ml phosphoramidon, leupeptin, aprotinin andchymostatin), test compound (or equivalent volume of solvent), 20 pM¹²⁵I-human eotaxin (2000 Ci/mmol), 25 ng unlabeled human eotaxin(non-specific binding wells only), and AML14.3D10 cells expressingeotaxin receptor cells, or eosinophils, are added sequentially in96-well, round-bottom, polystyrene plates to a final volume of 250 uL.Assay plates are then mixed and incubated for 60 minutes at 31° C. Afterincubation, assay plates are harvested onto Packard 96-well GF/CUnifilter plates treated with 0.33% polyethylenimine (PEI) using PackardFiltermate 196 cell harvester. Wells and filters are washed with 200 uL50 mM Hepes, pH 7.2 with 0.5M NaCl and 0.02% NaN₃. After filtration,GF/C plates are dried and sealed. 25 uL Packard Microscint-O scintillantare then added to each well and counted for 2 minutes on PackardTopcount (liquid ¹²⁵I setting).

EXAMPLE 8

Phosphoinositide 3-Kinase (PI-3K) Assay

AML14.3D10 expressing eotaxin receptor (CCCKR3) cells are incubated withtest compound and stimulated with eotaxin, RANTES, or MCP-3, pelletedand lysed in 1 mL lysis buffer (1% Nonidet P-40, 100 mM NaCl, 20 mMTris, pH 7.4, 10 mM iodoacetamide, 46 mM b-glyceraphosphate, 10 mM NaF,1 mM PMSF, 1 ug/mL leupeptin, 1 ug/mL chymostatin, 1 ug/mL antipain, 1ug/mL pepstatin A, and 1 mM sodium orthovanadate). Lysates are thenpre-cleared for 1 hr with uncoupled protein A Affi-Gel beads.Immunoprecipitation is then performed with p 85 polyclonal antiserum (1ul/mL lysate; Upstate Biologics, New York, N.Y.), coupled to protein AAffi-Gel beads (Bio-Rad) at 4° C. for 2 hr. Immunoprecipitates arewashed and subjected to in vitro lipid kinase assays by using a lipidmixture, 100 ul 0.1 mg/ml PtdIns and 0.1 mg/ml phosphatidylserinedispersed by sonication into solution in 20 mM HEPES, pH 7.0, and 1 mMEDTA. The reaction is initiated by the addition of 100 mM ATP and 20 uCi[gamma-³²P]ATP (3000 Ci/mmol) in 20 ul kinase buffer. The reaction isthen terminated after 15 min and the phosphoinositide lipids areseparated by thin layer chromatography (TLC) and visualized by exposureto iodine vapor autoradiography.

EXAMPLE 9

Chemotaxis Assay

AML14.3D10 expressing eotaxin receptor cells are isolated bycentrifugation (van Riper, G., et al. (1994)J. Immunol. 152, 4055-4061)for 15 min at 150×g, washed and resuspended at 10⁷ cells/ml in HBSS (pH7.4) containing 1 mM CaCl₂ and 1 mM MgCl₂ (chemotaxis buffer). Thechemotaxis experiments is then performed in Transwell dishes (6.5 mm,Costar, Cambridge, Mass.). The lower chamber contains 0.6 ml ofchemotaxis buffer and is separated from the upper chamber containing 10⁶cells by a 5-mm pore Nucleopore polycarbonate membrane (NucleoporeCorporation, Pleasanton, Calif.). After a 15 min preincubation at 37°C., test compound and eotaxin, RANTES, or MCP-3 are added to the lowerchamber to a final concentration of 300 nM. After 2 hrs at 37° C., theupper chamber inserts are removed, and the cells that migrate to thelower chamber are enumerated by a Coulter Counter (Coulter Electronics,Hialeah, Fla.).

EXAMPLE 10

Ligand-Dependent Inositol Phosphate Release Assay

AML14.3D10 expressing eotaxin receptor cells are labeled with [³H]inositol (10 uCi/ml) for 24 hrs as described (Wu, D., et al. (1993)Science 261, 101-103). Test compound and arious concentrations ofeotaxin, MCP-3, or RANTES are then added to the cells for 30 min. Thecells are lysed in 10% perchloric acid, neutralized in 2 N KOH andcentrifuged. The supernatant is transferred to columns containing 0.5 mlAG1-X8 anion exchange resin, washed with 6 ml borax buffer and elutedwith 0.3 ml formic acid (0.1 M). The eluted samples are mixed withscintillation cocktail and counted.

EXAMPLE 11

Acidification Rate Assay

AML14.3D10 expressing eotaxin receptor cells are subject to serumstarvation for 16 hrs. The cells are then mixed at a 3:1 (v/v) ratiowith low melting temperature agarose. A 10 ul drop of the cell/agarosemixture is pipetted into a sterile Capsule Cup (Molecular Devices) at acell density of approximately 200,000 cells/cup. The cell/agarose dropforms a gel after about 5 min, and is assembled into the cup between two3 um porosity polycarbonate membranes with running medium. The assembledcapsule cups is placed into the sensor chambers and then placed on theCytosensor Microphysiometer (Molecular Devices) containing 1 ml ofrunning medium. The chambers are allowed to equilibrate for 1 hr at 37°C. with a flow rate of 100 ul/min. The experiment is initiated with an 8min exposure of eotaxin, RANTES, MCP-3 and test compound at variousconcentrations and the acidification rate over baseline will be measuredin the medium (McConnell, H. (1992) Science 257, 1906-1912) until thecells return to the unstimulated level.

EXAMPLE 12

Actin Polymerization Assay

AML14.3D10 expressing eotaxin receptor cells are diluted in APA buffer(HBSS; 25 mM Hepes; 0.2% BSA, pH7.2) at a concentration of 4×10⁶/ml. Oneul of test compound and eotaxin, RANTES, or MCP-3 added into a 96-wellplate and incubated at 37° C. 100 ml of cells are added to the plate andincubated for 20 sec to which 100 ml of APA cocktail (2 mls 8%formaldehyde; 460 uLs 0.33 uM Rhodamine-phalloidin; 1.85 mg 200 ug/mllysolecithin; 7.25 mls HBSS) is added. Plates are then centrifuged at2000 RPM for 5 min, cleared and then 100 ul of HBSS is added to allwells which are read in a Fluoroskan II fluorometer.

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various adaptations, changes, modifications,substitutions, deletions, or additions of procedures and protocols maybe made without departing from the spirit and scope of the invention.For example, procedures other than the particular experimentalprocedures as set forth herein above may be applicable as a consequenceof degeneracy and variations in the sequences of the proteins and DNA ofthe invention indicated above. Likewise, the characterization dataobserved may vary slightly according to and depending upon theparticular assay or characterization method employed, and such expectedvariations or differences in the results are contemplated in accordancewith the objects and practices of the present invention. It is intended,therefore, that the invention be defined by the scope of the claimswhich follow and that such claims be interpreted as broadly as isreasonable.

1-35. (canceled)
 36. An isolated nucleic acid which encodes a humaneosinophil eotaxin receptor receptor, said nucleic acid having thenucleotide sequence as set forth in SEQ ID NO:2.
 37. The nucleic acid ofclaim 36 which further comprises the nucleotide sequence as set forth inSEQ ID NO:3.
 38. The nucleic acid of claim 36 which further comprisesthe nucleotide sequence as set forth in SEQ ID NO:4.
 39. A vectorcomprising a nucleic acid which encodes a human eosinophil eotaxinreceptor receptor, said nucleic acid having the nucleotide sequence asset forth in SEQ ID NO:2.
 40. The vector according to claim 39 which isselected from the group consisting of: plasmids, modified viruses, yeastartificial chromosomes, bacteriophages, cosmids and transposableelements.
 41. An isolated mammalian host cell comprising the vectoraccording to claim
 39. 42. The host cell of claim 41 which is from theAML14.3D10 cell line.
 43. A method to determine the presence of acompound which binds to a human eosinophil eotaxin receptor comprising:(a) introducing a nucleic acid which encodes the human eosinophileotaxin receptor which comprises the nucleotide sequence (SEQ ID NO:2)into a cell under conditions so that eosinophil eotaxin receptor isexpressed; (b) introducing a detector molecule or a nucleic acidencoding a detector molecule into the cell, wherein the detectormolecule is directly or indirectly responsive to a eosinophileotaxin-ligand binding event; (c) contacting the cell with a compoundsuspected of binding to the eosinophil eotaxin receptor; and (d)determining whether the compound binds to the eosinophil eotaxinreceptor by monitoring the detector molecule.
 44. The method of claim 43wherein the result of step (d) is compared to that obtained using aknown ligand of the eosinophil eotaxin receptor.
 45. The method of claim44 wherein the known ligand of the eosinophil eotaxin receptor iseotaxin.
 46. The method of claim 44 wherein the known ligand of theeosinophil eotaxin receptor is RANTES.
 47. The method of claim 44wherein the known ligand of the eosinophil eotaxin receptor is MCP-3.48. The method of claim 43 wherein the eosinophil eotaxin receptor isexpressed in a host cell which does not naturally express the humaneosinophil eotaxin receptor
 49. The method of claim 48 wherein the hostcell is from the AML14.3D10 cell line.